1,3-Benzenedicarboxylic acid, 5,5'-[1,2-ethanediylbis[thio-1,3,4-thiadiazole-5,2-diyl-2,1-diazenediyl[5-amino-3-(1,1-dimethylethyl)-1H-pyrazole-4,1-diyl]]]bis-, potassium salt (1:4)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 07 September 2011 and 27 September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

S196941

IUPAC Name:

S196941

Details on test material:

Sponsor's identification: S196941
Description : dark yellow/orange powder
Lot number : #NK1-24
Purity : 91.6% w/w
Date received: 16 March 2011
Expiry date : 31 March 2012
Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood wood flakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:

other: Please see below for Vehicle Determination Record

Concentration:

Each group was exposed to concentrations of 25%, 10% or 5% v/v 1% pluronic L92 in distilled water

No. of animals per dose:

Groups of four mice were treated

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test item at a concentration of 25% w/w in 1% pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.
Main Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.
3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.
Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

None provided.

Results and discussion

Positive control results:

Methods. A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a suspension in 1% pluronic L92 in distilled water at a concentration of 25% v/v. A further control group of five animals was treated with 1% pluronic L92 in distilled water alone.

Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration (% v/v) in
1% pluronic L92 in distilled water Stimulation Index Result
25 6.77 Positive

Conclusion. α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: Concentration (% w/w) in 1% pluronic L92 in distilled water Stimulation Index Result 5 0.98 Negative 10 1.08 Negative 25 0.96 Negative

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 4

Any other information on results incl. tables

 RESULTS

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3.

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the dose levels selected for the main test were 25%,10% and 5% w/w in1% pluronic L9 2in distilled water.

Table 1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (%w/w) in 1% pluronic L92in distilled water	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
25	S-1	18	18	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration (%w/w) in 1% pluronic L92in distilled water

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

25

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table 3              Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (%w/w) in 1% pluronic L92in distilled water	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
25	S-1	0.230	0.240	0.250	0.250	0.245	0.240
overall mean (mm)		0.235		0.250		0.243
overall mean ear thickness change (%)		na		6.383		3.191

na=     Not applicable

 Main Test

 Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in 1% pluronic L92in distilled water	Stimulation Index	Result
5	0.98	Negative
10	1.08	Negative
25	0.96	Negative

 Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Table 4              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (%w/w) in 1% pluronic L92in distilled water	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	6070.85	758.86	na	na
5	5949.80	743.73	0.98	Negative
10	6566.56	820.82	1.08	Negative
25	5846.46	730.81	0.96	Negative

dpm=  Disintegrations per minute

a=      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=      Stimulation Index of 3.0 or greater indicates a positive result

na =    Not applicable

Table 5              Individual Clinical Observations and Mortality Data

Concentration (% w/w) in 1% pluronic L92in distilled water	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
5	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
10	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
25	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Individual bodyweights and bodyweight changes for test and control animals are given in Table 6.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 6              Individual Bodyweights and Bodyweight Changes

Concentration (% w/w) in 1% pluronic L92in distilled water	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	18	19	1
	1-2	18	19	1
	1-3	19	20	1
	1-4	19	20	1
5	2-1	21	20	-1
	2-2	20	20	0
	2-3	17	18	1
	2-4	18	18	0
10	3-1	17	18	1
	3-2	19	20	1
	3-3	18	19	1
	3-4	18	18	0
25	4-1	19	20	1
	4-2	18	18	0
	4-3	17	17	0
	4-4	18	19	1

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the test.
The test item did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC or Regulation (EC) No 1272/2008. No symbol/risk phrase or signal word/hazard statement are required.

Executive summary:

Introduction. 

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed tobe compatible withthe following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted22 July 2010)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test item as asolution/suspensionin1% pluronic L92 in distilled waterat concentrations of 25%,10% or 5% w/w. A further group of four animals was treated with1% pluronic L92 in distilled wateralone.

Results. 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in 1% pluronic L92in distilled water	Stimulation Index	Result
5	0.98	Negative
10	1.08	Negative
25	0.96	Negative

Conclusion. 

The test item was considered to be anon-sensitiserunder the conditions of the test.

The test item did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC or Regulation (EC) No 1272/2008. No symbol/risk phrase or signal word/hazard statement are required.